Immunosuppressive protocols with tacrolimus after cryopreserved aortal allotransplantation in rats.

OBJECTIVES AND DESIGN:The aim of our study was to simulate in rats all aspects and techniques used in our new clinical program of cryopreserved alloarterial transplantation and investigate the influence of two immunosuppressive protocols with tacrolimus on acute rejection of these allografts. MATERIALS AND METHODS:Cryopreserved abdominal aortic grafts were transplanted between Brown-Norway and Lewis rats. Tacrolimus (0.2 mg/kg daily) was administered from day 1 to day 30 (TAC1) or from day 7 to day 30 (TAC7), respectively. No immunosuppressed isogeneic (ISO) and allogeneic (ALO) rats combination served as control. Aortal wall infiltration by immunocompetent cells (MHC II+ cells of recipient origin) was studied on day 30 after transplantation. Flow cytometry was used for the analysis of day 30 sera for the presence of donor specific anti-MHC class I and II antibodies. RESULTS:The aortal allografts in both immunosuppressed groups showed regular morphology of aortal wall with no depositions of immunoglobulin G on day 30. The adventitial infiltration of non-immunosuppressed aortal allografts by MHC class II positive cells of recipient origin was significantly higher (ALO 20.7±6.7 cells, P<0.001) compared to both immunosuppressed groups (TAC1 5.9±5.5 cells, TAC7 6.1±5.1 cells). Day 30 sera from the allogeneic non-immunosuppressed animals decreased significantly the binding of fluorescence-labelled MHC class I (46.9±19.4%) and class II (65.8±11.9%) antibody to donors spleen cells compared with day 30 sera from both immunosuppressed groups (TAC1, anti-MHC class I 102.4±4.2%, p < 0.001, anti-MHC class II 102.6±6.0%), (TAC7, anti-MHC class I 79.9±3.3%, p < 0.001, anti-MHC class II 80.9±2.7%). CONCLUSION:Both immunosuppressed protocols with tacrolimus (administration from day 1 or from day 7 following transplantation) were able to suppress acute cell- and antibody-mediated rejection of cryopreserved abdominal aortic allografts processed in accordance with our new standardized clinical protocol

Influence of the new standardized clinical cryopreservation/slow thawing protocol on immunogenicity of arterial allografts in rats.

OBJECTIVES AND DESIGN:At the present time there are two waiting list for patients with vascular prosthetic infection indicated for arterial transplantation in the Czech Republic. The inclusion of each patient for cold-stored or cryopreserved arterial transplantation is the preference of indicating surgeon. In this experimental work we studied the immunogenicity of rat aortal allografts treated by our new clinical cryopreservation/slow thawing protocol. MATERIAL AND METHODS:Brown-Norway (BN) (N = 6, 203-217 g) or Lewis (LEW) (N = 6, 248-254 g) abdominal aortal grafts treated in accordance with our new clinical cryopreservation/slow thawing protocol were orthotopically transplanted to Lewis recipients (N = 12, 191-245 g). Aortal wall histology and infiltration by recipient immune cells, as well as donor specific anti MHC class I and II antibodies in recipient serum were studied in both isografts and allografts on day 30 postransplant. Core data of cryopreserved allografts were compared to our previous data of cold-stored aortal allografts treated in accordance with our clinical cold-storage protocol. RESULTS:Cryopreserved allografts showed regular morphology of aortal wall with clear differentiation of all three basic anatomical layers on day 30 postransplant. Intimal layer showed no hyperplasia, luminal surface was covered by endothelial cells. No statistical difference was observed in tunica media thickness between isografts and allografts. The medial layer showed no necrosis, shrinkage or immunoglobuline G deposition in any experimental group. The adventitial infiltration by immune cells was significantly higher (P<0.05) in allografts. Cryopreserved allografts showed significant lower activation of both cell- and antibody mediated immunity compared to historical data of cold-stored allografts. CONCLUSION:Aortal wall histology of rat allografts treated by our new standardized clinical cryopreservation/slow thawing protocol was comparable to that of the cryopreserved isografts on day 30 posttranspant. The immunogenicity of cryopreserved aortal allografts was significantly lower compared to that of cold-stored aortal allografts

Immunosuppressive protocols with tacrolimus after cryopreserved aortal allotransplantation in rats

<div><p>Objectives and design</p><p>The aim of our study was to simulate in rats all aspects and techniques used in our new clinical program of cryopreserved alloarterial transplantation and investigate the influence of two immunosuppressive protocols with tacrolimus on acute rejection of these allografts.</p><p>Materials and methods</p><p>Cryopreserved abdominal aortic grafts were transplanted between Brown-Norway and Lewis rats. Tacrolimus (0.2 mg/kg daily) was administered from day 1 to day 30 (TAC1) or from day 7 to day 30 (TAC7), respectively. No immunosuppressed isogeneic (ISO) and allogeneic (ALO) rats combination served as control. Aortal wall infiltration by immunocompetent cells (MHC II+ cells of recipient origin) was studied on day 30 after transplantation. Flow cytometry was used for the analysis of day 30 sera for the presence of donor specific anti-MHC class I and II antibodies.</p><p>Results</p><p>The aortal allografts in both immunosuppressed groups showed regular morphology of aortal wall with no depositions of immunoglobulin G on day 30. The adventitial infiltration of non-immunosuppressed aortal allografts by MHC class II positive cells of recipient origin was significantly higher (ALO 20.7±6.7 cells, P<0.001) compared to both immunosuppressed groups (TAC1 5.9±5.5 cells, TAC7 6.1±5.1 cells). Day 30 sera from the allogeneic non-immunosuppressed animals decreased significantly the binding of fluorescence-labelled MHC class I (46.9±19.4%) and class II (65.8±11.9%) antibody to donors spleen cells compared with day 30 sera from both immunosuppressed groups (TAC1, anti-MHC class I 102.4±4.2%, <i>p</i> < 0.001, anti-MHC class II 102.6±6.0%), (TAC7, anti-MHC class I 79.9±3.3%, <i>p</i> < 0.001, anti-MHC class II 80.9±2.7%).</p><p>Conclusion</p><p>Both immunosuppressed protocols with tacrolimus (administration from day 1 or from day 7 following transplantation) were able to suppress acute cell- and antibody-mediated rejection of cryopreserved abdominal aortic allografts processed in accordance with our new standardized clinical protocol.</p></div

Anti MHC class II antibodies in serum.

<p>The percentage of binding of the anti-MHC class II antibody (anti-RT1.D, OX-17) to quiescent BN splenocytes in the presence of sera obtained from recipient rats pretransplant (POD 0) and on day 30 after transplantation (POD 30). ISO—isogeneic group with no immunosuppression. ALO—allogeneic group with no immunosuppression. TAC1—from day 1 immunosuppressed allogeneic group. TAC7—from day 7 immunosuppressed allogeneic group.</p

Anti MHC class I antibodies in serum.

<p>The percentage of binding of the anti-MHC class I antibody (anti-RT1.Ac, OX-27) to quiescent BN splenocytes in the presence of sera obtained from recipient rats pretransplant (POD 0) and on day 30 after transplantation (POD 30). ISO—isogeneic group with no immunosuppression. ALO—allogeneic group with no immunosuppression. TAC1—from day 1 immunosuppressed allogeneic group. TAC7—from day 7 immunosuppressed allogeneic group.</p

Weight increase on day 30 after transplantation of aortic grafts expressed as a percentage of preoperative weight.

<p>No immunosuppressed animals (group ISO, group ALO) showed significantly higher weight increase on day 30 when compared to from day 1 immunosuppressed animals (group TAC1). However, the weight increase in from day 7 immunosuppressed animals (group TAC7) was compared to animals without immunosuppression (group ISO, group ALO).</p

Assessments of rat cryopreserved aortic graft on day 30 after transplantation into the abdominal aorta.

<p>Assessments of rat cryopreserved aortic graft on day 30 after transplantation into the abdominal aorta.</p

a1, a2, b1, b2. Representative light microscopic histological features of adventitial infiltration of cryopreserved allografts by mononuclear cell at 30 days following transplantation.

<p>The adventitial infiltration of Brown-Norway cryopreserved aortal grafts by MHC class II positive cell of Lewis origin (stained brown) (Fig 3 a1) was significantly reduced by both types of immunosuppressive protocols with tacrolimus (Fig 3 a2). The adventitial infiltration of Brown-Norway aortal grafts by CD4+ cell (Fig 3 b1) was significantly reduced by both immunosuppressive protocols with tacrolimus as well (Fig 3 b2). Original magnification ×400.</p